A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for in situ and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface. Human mucin1 protein (MUC1) aptamer was immobilized onto AuNPs for capturing MUC1-positive MCF-7 cells. Au@luminol and CdS QDs as ECL nanoprobes were covalently linked with concanavalin A (ConA) and epidermal growth factor (EGF) to label MCF-7 cells on the two areas of the cytosensor separately. Compared to conventional multiplex biosensor, we demonstrated a novel analysis platform for the simultaneous detection of multiple cell-surface receptors; it could provide two sensitive and potential-resolved ECL signals during one potential scanning and avoid cross-reactivity between the two nanoprobes. The quantification of MCF-7 cells on the two spatially resolved areas could be achieved over the linear range from 102 to 1.0 × 106 cells mL-1 with a detection limit of 20 cells mL-1. This multiplex cytosensor was further applied for simultaneous quantitative evaluation of the expression levels of mannose and EGFR on MCF-7 cells, revealed that the average numbers of mannose and EGFR per captured MCF-7 cell were 1.2 × 106 and 0.86 × 105 with the relative standard deviation of 5.3% and 4.2%, respectively. The multiplex cytosensor was capable of evaluating multiple cell-surface receptors, which would be beneficial to developing a better diagnostic tool for diseases.
Keywords: cell-surface receptors; dual signal system; epidermal growth factor receptor; mannose; multiplex cytosensor.